Zika virus (ZIKV) is a vertically and sexually transmissible virus resulting in severe congenital malformation. The goal of this study was to develop an ovine model of ZIKV infection. Between 28–35 days gestation (DG), four pregnant animals were infected with two doses of 6 × 106 PFU of ZIKV; four control animals received PBS. Animals were evaluated for 45 days (D) post-infection (PI) and necropsies were performed. Viral RNA was detected in infected ewe peripheral blood mononuclear cells (PBMC) during the first week PI; however, all fluids and tissues were negative upon culture. Anti-ZIKV IgM (1:400) and neutralizing antibodies were detected in all infected animals. Clinical disease, virus, or ZIKV antibodies were not detected in control ewes. After two weeks PI, fetal loss occurred in two infected animals, and at necropsy, three infected animals had placental petechiation and ecchymosis and one had hydramnion. Fetal morphometrics revealed smaller cranial circumference to crown-rump length ratios (p < 0.001) and relative brain weights (p = 0.038) in fetuses of infected animals compared with control fetuses. Immunophenotyping indicated an increase in B cells (p = 0.012) in infected sheep. Additionally, in vitro experiments using both adult and fetal cell lines demonstrated that ovine cells are highly permissive to ZIKV infection. In conclusion, ZIKV infection of pregnant sheep results in a change in fetal growth and gestational outcomes.

Zika virus (ZIKV) is an arbovirus that causes birth defects, persistent male infection, and sexual transmission in humans. The purpose of this study was to continue the development of an ovine ZIKV infection model; thus, two experiments were undertaken. In the first experiment, we built on previous pregnant sheep experiments by developing a mid-gestation model of ZIKV infection. Four pregnant sheep were challenged with ZIKV at 57-64 days gestation; two animals served as controls. After 13-15 days (corresponding with 70-79 days of gestation), one control and two infected animals were euthanized; the remaining animals were euthanized at 20-22 days post-infection (corresponding with 77-86 days of gestation). In the second experiment, six sexually mature, intact, male sheep were challenged with ZIKV and two animals served as controls. Infected animals were serially euthanized on days 2-6 and day 9 post-infection with the goal of isolating ZIKV from the male reproductive tract. In the mid-gestation study, virus was detected in maternal placenta and spleen, and in fetal organs, including the brains, spleens/liver, and umbilicus of infected fetuses. Fetuses from infected animals had visibly misshapen heads and morphometrics revealed significantly smaller head sizes in infected fetuses when compared to controls. Placental pathology was evident in infected dams. In the male experiment, ZIKV was detected in the spleen, liver, testes/epididymides, and accessory sex glands of infected animals. Results from both experiments indicate that mid-gestation ewes can be infected with ZIKV with subsequent disruption of fetal development and that intact male sheep are susceptible to ZIKV infection and viral dissemination and replication occurs in highly vascular tissues (including those of the male reproductive tract).Viruses 2020, 12, 291 2 of 23 and negative gestational outcomes when infection occurs during pregnancy. In humans, ZIKV is believed to result in trimester-specific effects to the fetus [2][3][4][5][6][7]. Established animal models of ZIKV infection during pregnancy that result in vertical transmission include non-human primates (NHP) and type-1 interferon/interferon receptor deficient mice. In NHP models, first trimester infection most frequently results in fetal demise and reduced fetal development even in asymptomatic mothers, while second trimester infections tend to produce fetuses with higher viral loads but greater fetal viability [8][9][10]. In all NHP models, fetal pathology ranges from mild to severe manifestations, consistent with Congenital Zika Syndrome (CZS) seen in humans [11][12][13][14][15][16]. While NHP models of ZIKV infection during pregnancy recapitulate human infection, other outbred host models can serve as surrogates when ethical and economic constraints create obstacles to the use of NHP.Sheep offer a unique animal model for translational biomedical research, and have long been used as a model for human pregnancy and fetal development due to longer gestation and comparably staged rates of fetal d...

Zika virus (ZIKV) circulates as two separate lineages, with significant genetic variability between strains. Strain-dependent activity has been reported for dengue virus, herpes simplex virus and influenza. Strain-dependent activity of subject specimens to a virus could be an impediment to serological diagnosis and vaccine development. In order to determine whether ZIKV exhibits strain-dependent activity when exposed to antibodies, we measured the neutralizing properties of polyclonal serum and three monoclonal antibodies (ZKA185, 753(3)C10, and 4G2) against three strains of ZIKV (MR−766, PRVABC59, and R103454). Here, MR−766 was inhibited almost 60% less by ZKA185 than PRVABC59 and R103454 (p = 0.008). ZKA185 enhanced dengue 4 infection up to 50% (p = 0.0058). PRVABC59 was not inhibited by mAb 753(3)C10 while MR−766 and R103453 were inhibited up to 90% (p = 0.04 and 0.036, respectively). Patient serum, regardless of exposure history, neutralized MR−766~30%−40% better than PRVABC56 or R103454 (p = 0.005−0.00007). The most troubling finding was the significant neutralization of MR−766 by patients with no ZIKV exposure. We also evaluated ZIKV antibody cross reactivity with various flaviviruses and found that more patients developed cross-reactive antibodies to Japanese encephalitis virus than the dengue viruses. The data here show that serological diagnosis of ZIKV is complicated and that qualitative neutralization assays cannot discriminate between flaviviruses.Trop. Med. Infect. Dis. 2020, 5, 38 2 of 14 with ZIKV, dengue virus (DENV) and other endemic flaviviruses is recommended by the Centers for Disease Control and Prevention ( CDC) to confirm diagnosis when RT-PCR is negative, and IgM ELISA is "not negative" [7]. Unfortunately, even PRNT can be problematic for diagnosis with over half of patients exhibiting significant neutralization of ZIKV, DENV, and other flaviviruses when positive for ZIKV IgM [9].ZIKV co-circulates with other flaviviruses, including DENV, West Nile virus (WNV), Japanese Encephalitis virus (JEV) and Yellow Fever virus (YFV). Antibody-based assays can be problematic as serological cross-reactivity can confound results [10][11][12]. Furthermore, previous exposures and co-infections can further complicate diagnostic tests [13][14][15]. Additional difficulties arise when diagnosing clinical samples in different locations as reference virus and antigen sources can produce divergent results in response to locally circulating viral isolates [16].Prior to the 2016 outbreak, there were very few ZIKV isolates available for use, which included the prototype MR−766 isolate as well as IbH 30656 a Nigerian isolate and DAK AR 41524 from Senegal (BEIResources.org). MR−766 had been extensively characterized prior to the outbreak and was the reference strain used by many researchers in the early days following emergence [6,[17][18][19][20]. Within a year of emergence, the CDC prototype PRVABC59 was released for use through BEI resources and many labs began using this modern isolate for their research and dia...

Zika virus (ZIKV) co-circulates with several closely related flaviviruses which exhibit similar clinical manifestations thus, clinicians rely on molecular and serological techniques for diagnosis. Cross-reactivity of patient specimens to flaviviruses is a significant impediment to serological diagnosis in areas where multiple flaviviruses co-circulate. Furthermore, patient exposure history to any of these viruses could complicate serological response patterns which could result in over and/or underdiagnosis of ZIKV infection. Three strains of ZIKV, dengue serotypes 1-4, West Nile virus, Japanese Encephalitis virus, and Yellow Fever virus were evaluated for neutralizing properties against 3 monoclonal antibodies, 4 ZIKV-na&iuml;ve patients with flavivirus exposure history, 5 patients with verified ZIKV exposure and unknown flavivirus exposure history, and 5 flavivirus-naive patients with ZIKV-only exposure. Patients na&iuml;ve for ZIKV exposure effectively neutralized multiple strains of ZIKV. Overall, the prototype ZIKV isolate MR-766 did not behave like the other ZIKV isolated used in this study. MR-766 was neutralized more completely by polyclonal patient serum than recent ZIKV isolates. MR-766 was neutralized better than dengue virus in ZIKV-na&iuml;ve patients with prior dengue exposure. MR-766 was neutralized significantly less than recent ZIKV isolates when treated with monoclonal antibodies. The data herein show that without RT-PCR, serological diagnosis may not be possible in areas where multiple flaviviruses are endemic.

West Nile virus (WNV) neuroinvasive disease threatens the health and well-being of horses and humans worldwide. Disease in horses and humans is remarkably similar. The occurrence of WNV disease in these mammalian hosts has geographic overlap with shared macroscale and microscale drivers of risk. Importantly, intrahost virus dynamics, the evolution of the antibody response, and clinicopathology are similar. The goal of this review is to provide a comparison of WNV infection in humans and horses and to identify similarities that can be exploited to enhance surveillance methods for the early detection of WNV neuroinvasive disease.